The contractile elements of cardiac and skeletal muscle are critically dependent on the intracellular concentration of calcium ion, for the regulation of their contractile state. In turn the intracellular concentration of calcium ion is controlled by membrane systems which can be isolated and display in vitro Ca ion transport properties. It is the aim of this research proposal to combine physiological, physical and chemical methods to investigate the mechanisms of Ca ion uptake and release by these membranes, and define the sensitivity of the contractile system to such control. The proposed work involves isolation of various intracellular membrane fractions, characterization of their origin within the subcellular structure, description of ultrastructural details in terms of molecular arrangement within the membrane bilayer, definition of transport and enzyme kinetics in transient (milliseconds) and steady states, study of macromolecular conformation in relation to enzyme intermediate states, isolation and purification of proteins and tryptic fragments, chemical modification and labeling of enzymatic sites, experiments with lipid bilayers and reconstituted membranes, monitoring of sarcomere length and contractile parameters under controlled sarcomere length. Pathologic conditions and pharmacologic effects will be investigated in the light of the parameters described above.